322 Single-cell RNA sequencing of afamitresgene autoleucel indicates mechanisms of persistence and anti-tumor activity

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Background Afamitresgene autoleucel (afami-cel) is an engineered T-cell receptor (TCR) T-cell therapy targeting tumors expressing melanoma-associated antigen (MAGE) A4 and is the first such therapy with advanced FDA approval for a solid tumor. In the Phase 2 SPEARHEAD-1 trial in patients with advanced synovial sarcoma or myxoid (with/without round cell) liposarcoma, greater long-term afami-cel pharmacokinetic exposure was associated with clinical benefit. 1 We explored gene expression profiles and functional characteristics of afami-cel manufactured product (MP, i.e., before infusion into patients) associated with long-term persistence.Methods Afami-cel MP were flow sorted into transduced (t)CD4+ and tCD8+ subpopulations. Single-cell RNA sequencing was performed on afami-cel tCD4+ and tCD8+ MP subpopulations from 22 and 23 patients, respectively, using 10X Genomics single-cell 5’ technology. In vitro cytotoxicity assays measured killing of a target HLA-A2/MAGE-A4-expressing tumor cell line over 125 hours co-culture with afami-cel MP subpopulations. qPCR specific to the engineered TCR measured afami-cel persistence in patient peripheral blood, with pharmacokinetic exposure calculated as area under the curve over 0–6 months after infusion.Results We saw distinct gene expression profiles associated with greater pharmacokinetic exposure in patients. In tCD8+ MP samples, this was characterized by an effector function and long-lived memory formation expression profile, whereas expression of genes involved in T-cell dysfunction was associated with lower pharmacokinetic exposure. In tCD4+ MP samples, higher exposure was associated with expression of T-cell activation genes and lower exposure was associated with pro-apoptotic, anti-proliferative gene expression, including genes shared with the tCD8+ T-cell dysfunction-like profile. Afami-cel tCD8+ MP samples demonstrated strong cytotoxicity in vitro and sustained cytotoxic activity was associated with greater long-term pharmacokinetic exposure. Loss of initial cytotoxic activity was associated with a higher percentage of cells expressing a T-cell dysfunction-like gene profile. Afami-cel tCD4+ MP samples also displayed in vitro cytotoxic activity, and greater in vitro cytotoxicity of afami-cel tCD4+ T-cells was associated with CD4+ T-cell activation and effector gene expression.Conclusions We hypothesize that MP with T-cell subsets poised for sustained effector function and long-lived memory formation are capable of longer post-infusion persistence, sustaining higher afami-cel pharmacokinetic exposure, and will discuss the gene expression programs underlying these different cellular potentials.Acknowledgements This study was sponsored by Adaptimmune. Writing/editorial support by Christine Ingleby, DPhil, CMPP, Envision Pharma Inc. (Glasgow, UK) funded by Adaptimmune. The authors would like to thank the patients, their families, caregivers, and the teams involved in the SPEARHEAD-1 trial.Trial Registration SPEARHEAD-1 is registered at ClinicalTrials.gov with ID NCT04044768.Reference D’Angelo SP, Araujo DM, Abdul Razak AR, et al. Afamitresgene autoleucel for advanced synovial sarcoma and myxoid round cell liposarcoma (SPEARHEAD-1): an international, open-label, phase 2 trial. Lancet. 2024; 403: 1460–1471.Ethics Approval SPEARHEAD-1 was approved by local or independent institutional review boards or ethics committees at each participating site and all patients provided written informed consent.

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